Mechanism Of Hydrogen Sulfide Preconditioning Of Mesenchymal Stem Cell Derived Extracellular Vesicles In Hypoxic-ischemic Brain Injury

BackgroundHypoxic-ischemic encephalopathy(HIE)in neonates is a major disease that results from severe perinatal asphyxia,often resulting in neonatal death and long-term neurological sequelae.HIE incidence is high and mortality is high,survivors often suffer from long-term neurological dysfunction.There is no perfect and unified clinical treatment strategy for neonate HIE,and the current treatment is mainly comprehensive supportive treatment,but its therapeutic effect is not very significant,so it is particularly important to seek a safe and effective treatment strategy.After hypoxic-ischemia(HI),changes in cerebral blood flow caused energy metabolism disorders in nerve cells.The release of a large number of excitatory amino acids,oxygen free radicals,nitric oxide and proinflammatory mediators released by apoptotic cells to activate microglial lead to the changes in the immune microenvironment of the Central nervous system(CNS).The destruction of the blood-brain barrier after HI and the change of the immune microenvironment in CNS induced peripheral immune cells to infiltrate into CNS.Activated microglia/mononuclear-macrophages have different functional phenotypes,M1 microglia/mononuclear-macrophages release pro-inflammatory factors,further aggravating neuroinflammation and tissue damage;M2 microglia/mononuclear macrophages secrete anti-inflammatory factors and neurotrophic factors,which participate in the anti-inflammatory process to promote tissue repair.Therefore,inhibiting the activation of microglia after HI and promoting the M2 polarization of microglia/mononuclear-macrophage has great significance for inhibiting neuroinflammation and reducing brain damage.Extracellular vehicles(EVs)are spherical,membranous vesicles surrounded by lipid bilayer.As the third signal transduction pathway after cell contact dependence and cytokine signal transduction pathway,EVs can negatively regulate the genes of target cells through the rich miRNA it’s carries.EVs derived from Mesenchymal stem cell(MSCs)has a variety of biological effects,and can participate in the pathophysiological process of a variety of diseases through immune regulation,anti-apoptosis,anti-oxidation,and proliferation promotion.Hydrogen sulfide(H2S)is a kind of endogenous signal molecules involved in the regulation of a series of pathophysiological process of brain and nerve protection role in CNS,in many pathological processes,H2S can play the role of anti-inflammatory antioxidants curb inflammation.ObjectiveThe purpose of this study was to investigate whether H2S-EVs obtained from H2S modified MSCs and EVs have superior neuroprotective effects.At the same time,the possible mechanism of its neuroprotective effect was studied.Methods1.Establishment the Model:this study used C57BL/6J newborn mice 7 days after birth to establish HI model,and referred to Rice-Vannucci method for modeling.In the experimental group,the skin of the neck was cut open after anesthesia and fixed,and the right common carotid artery was dissociated and ligated.After returning to the side of the body of the female mice for 30 min,the mice were continuously anoxic at 8%O2+92%N2 and 37℃ for 2 h.The control group(Sham)only dissociated the right common carotid artery without ligation2.Experimental design2.1 Experimental design 1The effects of EVs and H2S-EVs on acute injury were evaluated at 72 h following HI.Mouse pups were randomly allocated into 4 groups:(1)Sham-treated with vehicle(PBS),(2)HI-treated with vehicle(PBS)(HI)(3)HI-treated with EVs(HI+EVs),(4)HI-treated with H2S-EVs(HI+H2S-EVs).A total of 100 μg EVs or H2S-EVs dissolved in 50 μl PBS were injected intracardially at 24 h following HI insult.The dose of EVs and H2S-EVs chosen was based on preliminary data from our laboratory.Mice were euthanized at 72 h following HI(PND 10)for further analysis.2.2.Experimental design 2The long-term effects of EVs and H2S-EVs were evaluated at 5-week after HI.Mouse pups were randomly allocated into the 4 groups as described in Experimental design 1.Neurobehavioral tests,as performed at this 5-week post-HI,included the Novel object recognition test and Morris water maze test.Following behavioral testing,animals were euthanized for assessment of brain atrophy.2.3.Experimental design 3The purpose of this experiment was to confirm that the neuroprotective effects associated with H2S-EVs were mediated by the EVs delivery of miR-7b-5p.To accomplish this goal,animals in the HI group were injected with PBS,H2S-EVs,H2S-EVs pretreated with the miR-7b-5p inhibitor(miR-7binhibitor),or its negative control(miR-7bINC)following HI exposure.The animals were then randomly divided into 4 groups:(1)HI+Vehicle(HI+Veh),(2)HI+H2S-EVs,(3)HI+H2S-EVs miR-7b-5p inhibitor control(HI+H2S-EVs-miR-7bINC),(4)HI+H2S-EVs miR-7b-5p inhibitor(HH+H2S-EVs-miR-7binhibitor).All EVs or vehicle treatments consisted of an intracardial injection administered at 24 h following HI insult.At 72 h following HI,edema,tissue loss and neuroinflamation were evaluated.2.4.Experimental design 4The purpose of this experiment was to assess whether the miR-7b-5p/FOS signaling pathway is involved in H2S-EVs-mediated neuroprotection in HI.Mice were treated with LV-mediated FOS siRNA(LV-FOS)and LV-mediated FOS siRNA negative control(LV-NC)at 72 h prior to HI exposure.Animals were then randomly divided into 4 groups:(1)HI+Vehicle(HI+Veh),(2)HI+H2S-EVs,(3)HI+LV-NC,(4)HI+LV-FOS.At 72 h following HI,edema,tissue loss and neuroinflammation were evaluated2.5.Experimental design 5The purpose of this experiment was to assess whether the miR-7b-5p has the neuroprotective effects in HI.Mice were treated with miR-7b-5p agomir and its negative control(NC)at 2 h prior to HI exposure.Animals were then randomly divided into 4 groups:(1)HI+Vehicle(HI+Veh),(2)HI+H2S-EVs,(3)HI+miR-7bNC,(4)HI+miR-7bagomir.At 72 h following HI,edema and tissue loss were evaluated3.Detection methods(1)Both EVs was identified by transmission electron microscopy,qNano and Western blot.(2)Immunofluorescence was used to observe the distribution of H2S-EVs in the brain tissue of 72 h after HI and in the primary microglia/BV-2 cells after Lipopolysaccharides(LPS)stimulation.(3)Western blot,Reverse transcription and real-time fluorescence quantitative real-time PCR(qRT-PCR),Flow Cytometry(FCM),immunohistochemistry,brain water content detection,TTC staining and other experiments were used to detect the expression levels of inflammatory factors and the activation degree of microglia in different experimental designs,and edema,cerebral infarction,and microglia/mononuclear-macrophage polarization phenotypes.(4)Behavioral test:five weeks after HI,new object recognition(NORT)and water maze were(MWM)used to detect the long-term neurological recovery of different groups of mice.4.Statistical analysisThe SPSS software program was used to perform statistical analyses.All values presented are expressed as the mean ± standard deviation.Daily performance scores in the MWM tests were evaluated with use of a repeated-measures two-way analysis of variance(ANOVA)with "days" as the within-subject factor and "groups" as the between-subject factor.Daily scores for the different groups in the MWM tests were compared using one-way ANOVA,following by the post hoc Bonferroni corrections test for multiple comparisons.Other data were also subjected to one-way ANOVA,followed by Bonferroni corrections or Dunnet’s tests for multiple post-hoc comparisons of means.Statistical significance was defined as p ＜0.05.Results1.Characteristics and content identification of both EVsBoth EVs were similar with regard to their protein marker expressions(CD9,TSG101)and sizes.Calnexin is expressed in MSCs but rarely in EVs.As based on TEM images,the isolated vesicles displayed a cup-shaped canonical morphology.2.Both EVs distribution and targets in vivo and in vitroPKH67-labeled H2S-EVs were also found to be localized in the Iba-1+microglia in ipsilateral lesion regions,but not in non-injured regions of the ipsilateral hemisphere or in contralateral hemispheres.And a relatively number of labeled H2S-EVs were also found in NeuN+neuronal cells and glial fibrillary acidic protein(GFAP)+astrocytes in lesion regions of the ipsilateral hemisphere.Analysis of H2S-EVs uptake as performed in primary microglia cultures revealed that labeled H2S-EVs began to accumulate in cells at 2 h following treatment Moreover,PKH67-labeled H2S-EVs were found to be localized in the perinuclear,intranuclear and cytoplasm regions of the cell.EVs accumulation was also observed within BV-2 cells.3.H2S-EVs treatment alleviated brain damage at 72 h following HIH2S-EVs treated animals demonstrated significantly less water content compared to the HI group(p＜0.01).Significantly reduced infarct volumes were observed in H2S-EVs treated animals as compared to both the HI(p＜0.001)and EVs(p＜0.05)groups as revealed from TTC staining.4.H2S-EVs treatment alleviated apoptosis at 72 h following HIResults from TUNEL staining indicated that significantly decreased amounts of cell apoptosis were present in the H2S-EVs as compared to the HI(p＜0.001)and EVs(p＜0.05)groups.H2S-EVs treated animals also showed significant reductions in cleaved caspase-3 levels as compared to both the HI(p＜0.001)and EVs(p＜0.05)groups5.Immunologic suppression of H2S-EVs treatment at 72 h following HIAs EVs have been reported to mediate immune responses,we next examined neuroinflammation responses following EVs treatment.Results from Iba-1 staining revealed that both EVs(p＜0.001)and H2S-EVs(p＜0.001)blocked HI-induced microglial activation.When comparing the effects of H2S-EVs versus EVs treatment,we found that the former showed significantly lower numbers of Iba-1+cells(p＜0.01).H2S-EVs treatment significantly attenuated levels of pro-inflammatory markers and pro-inflammatory cytokines within the ipsilateral cortex as compared with that of the HI group,including pro-inflammatory markers:CD11b(p ＜0.001),CD32(p＜0.001),CD86(p＜0.001),COX2(p＜0.001),iNOS(p ＜0.001)and pro-inflammatory cytokines:IL-1β(p＜0.001),IL-6(p ＜0.001),and TNFα(p＜0.001).H2S-EVs treatment also resulted in significantly greater amounts of immunologic suppression as compared with that of the EVs group for the following pro-inflammatory markers CDllb(p＜0.01),CD32(p＜0.001);CD86(p＜0.001)and pro-inflammatory cytokines IL-1β(p＜0.05).We also found that mRNA levels of CD206 were significantly increased in the H2S-EVs treatment group as compared with that of both the HI(p＜0.01)and EVs(p＜0.001)groupsFlow cytometry was used to separate populations of resident microglia(CD11b+/CD45low)and infiltrating immune cells(CD11b+/CD45high).As expected,CD11b+/CD45low accounted for the majority of CD11b+immune cells,while CD11b+/CD45high cells represented only 0.57%of CD11b+immune cells in the brain.The percent of CD11b+/CD45low cells within the ipsilateral cortex of the HI group(p＜0.001)was significantly lower than that found in the right cortex of the Sham group However,the percent of CD11b+/CD45high cells was significantly increased(p ＜0.001)in the HI versus Sham groupTo assess this possibility the ipsilateral cortex was subjected to flow cytometry for determining the ratios of M1-and M2-types in response to HI.Following HI,the percent of M1 monocytes/macrophages was significantly increased(p＜0.001),while M2 monocytes/macrophages significantly decreased(p＜0.01)as compared with the Sham group.The resulting ratio of M1/M2 monocytes/macrophages was significantly skewed(p<0.001)toward the M1 phenotype following HI insult as compared with that observed in the Sham group.In mice treated with H2S-EVs,the percent of M1 monocytes/macrophages showed a slight,but non-significant,decrease(p>0.05).Notably,H2S-EVs treatment significantly increased the percent of M2 monocytes/macrophages as compared with that obtained in both the HI(p<0.01)and EVs(p<0.01)groups.H2S-EVs treatment significantly promoted the resulting ratio of M1/M2 monocytes/macrophages to skew them toward the anti-inflammatory M2 phenotype as compared with that observed in the HI(p<0.001)and EVs(p<0.05)groups.HI increased the percent of M1 microglia(p<0.001)as compared with the Sham group,while the percent of M2 microglia was slightly,but non-significantly,increased(p>0.05).The resulting ratio of M1/M2 microglia was significantly skewed(p<0.001)toward the M1 phenotype following HI insult.A similar pattern of polarization was observed for microglia following H2S-EVs treatment.In these H2S-EVs treated mice the percent of M1 microglia was significantly decreased as compared with the vehicle treated group(p<0.001),while the percent of M2 microglia exhibited a similar response as that observed with the HI group(p>0.05)in response to HI.H2S-EVs treatment resulted in a significant decrease in the M1/M2 microglia ratio as compared with both the vehicle-treated(p<0.001)and EVs-treated(p<0.05)groups following HI insult 6.Neural functional recovery of H2S-EVs treatment in acute HI injuryTo assess the long-term consequences of postnatal EVs treatment on neural function,we examined the memory function in these mice at 42 days of age.The HI group failed to discriminate between the novel and familiar object and showed significant reductions in discrimination ratios as compared with that of the Sham group(p<0.05).A significant increase in discrimination ratios was obtained in the H2S-EVs as compared to that of the HI group(p<0.05).While,the EVs-treated group showed similar discrimination ratio as those in the HI group(p>0.05).Significant increases in escape latencies were present in the HI group on day 5,as compared with that of the Sham group(p<0.01),while H2S-EVs-treated animals showed significant reductions in escape latencies on the day 5(p<0.05)compared to the HI group.The EVs-treated group showed similar escape latencies as those in the HI group on all days of testing.On day 6,the number of crossings of the original platform location,the initial arrival time at the original platform location and the time spent in the quadrant target area were all measured.HI mice showed significant reductions in the number of crossings(p＜0.01)and quadrant target time(p ＜0.01),as well as spending significantly longer times searching for the original platform(p ＜0.01)as compared to the Sham group.However,H2S-EVs treated mice showed significant increases in the number of crossings(p＜0.05)and decreases in escape times(p＜0.01),but no differences in time spent in the quadrant target(p＞0.05)as compared to the HI group7.Up-regulated miR-7b-5p expression of H2S-EVs treatment plays a neuroprotective role at 72 h following HIAs results from most studies have indicated that EVs influence the fate of target cells mainly through their miRNA content,we next performed a miRNA sequencing analysis of these two types of EVs to determine whether miRNAs may potentially mediate the anti-inflammatory effects of EVs.H2S preconditioning up-regulated 19 miRNAs and down-regulated 7 miRNAs in EVs as compared to that observed with vehicle treatment.Among these,miR-7b-5p represented the maximally up-regulated miRNA enriched in response to H2S-EVs,and may thus be primarily responsible for the anti-inflammatory properties observed.We confirmed that NaHS treatment of MSCs remarkably up-regulated miR-7b-5p expression in EVs using qRT-PCRIn addition,we found that miR-7b-5p levels within the ipsilateral cortex were significantly decreased at 2 h,12 h,24 h,and 72 h post-HI(p＜0.001;p＜0.001;p＜0.001;p＜0.001;respectively).Both EVs treatment significantly increased miR-7b-5p levels as compared with that of untreated HI mice at 72 h post-HI(p＜0.001,for both).Notably,H2S-EVs treatment significantly increased miR-7b-5p expression as compared with that of the EVs-treated group(p ＜0.001).Results from fluorescent in situ hybridization(FISH)showed that miR-7b-5p was localized within Iba-1 microglia in the ipsilateral hemisphere at 72 h following HI Moreover,we confirmed the presence of miR-7b-5p in these H2S-EVs receiving the miR-7b-5p inhibitor,or its negative control,as achieved with use of qRT-PCR.Specifically,the miR-7b-5p inhibitor significantly decreased miR-7b-5p expression in H2S-EVs(p<0.001).To examine the potential role of miR-7b-5p in EVs as providing the beneficial effects of H2S-EVs in HI mice,mice received H2S-EVs derived from MSCs that had been pretreated with PBS,the miR-7b-5p inhibitor(H2S-EVs-miR-7binhibitor)or its negative control(H2S-EVs-miR-7bINC).Treatment with the H2S-EVs-miR-7binhibitor failed to produce similar effects on brain injury as that obtained in response to that of H2S-EVs or H2S-EVs-miR-7bINC administration.Edema and infarct volumes a were all significantly increased in the HI+H2S-EVs-miR-7binhibitor as compared with that in the HI+H2S-EVs-miR-7bINC group.Moreover,edema and infarct volumes in the HI+H2S-EVs-miR-7bINC group were reduced as compared with that in the HI group.We also found that miR-7b-5p agomir treatment(i.c.v)exerted similar effects on brain injury as that observed with H2S-EVs following HI insult,as evidenced from results obtained with edema and infarct volume analyses.We then investigated the effect of miR-7b-5p on neuroinflammation following HI.H2S-EVs-miR-7binhibitor significantly enhanced microglial activation compared with that of the HI+H2S-EVs-miR-7bINC group as assessed with Iba-1 staining analysis(P＜0.001).Results of qRT-PCR revealed that the H2S-EVs-miR-7binhibitor significantly suppressed the anti-inflammatory effects of H2S-EVs upon:CD16(p＜0.001),CD86(p＜0.001),IL-1β(p＜0.01)and TNFa(p＜0.001),while CD206 expression was significantly increased(p＜0.001)following HI.8.miR-7b-5p/FOS mediates the anti-inflammatory effects of H2S-EVs in acute HI injurymiR-7b-5p can bind to the FOS gene 3’-UTR as based on results obtained from computational and bioinformatics analysis using TargetScan.To confirm that a direct interaction was present between miR-7b-5p and FOS,we employed a luciferase reporter assay.With use of this assay,we found that luciferase activity of FOS was markedly decreased when transfected with miR-7b-5p mimics as compared with that observed in negative controls(p<0.001).As expected,we found that that H2S-EVs treatment significantly decreased levels of FOS mRNA(p<0.001)and c-FOS protein(p<0.01)compared with that observed in the HI alone group.Inhibition of miR-7b-5p reversed this EVs-induced reduction of FOS mRNA(p<0.001)and c-FOS protein(p<0.05)expression following HI.To assess whether FOS signaling is involved in H2S-EVs-mediated neuroprotection in response to HI,mice were treated with the LV-shFOS(injected i.c.v.)to silence FOS expression.Inhibition of FOS was verified with use of qRT-PCR and Western blotting assays.The down-regulated expression of FOS,as achieved with LV-shFOS,partially blocked HI-induced edema(p＜0.01)and infarct volumes(p＜0.001).Moreover,this LV-shFOS treatment reversed HI-induced microglial activation as assessed with Iba-1 staining(p<0.01)and pro-inflammatory genes expressions as assessed with qRT-PCR analysis,similar to that observed in response to H2S-EVs treatment.Conclusion and significanceOur study shows that H2S-EVs obtained by H2S modified MSCs has superior neuroprotective effect than EVs and we also confirmed the possible mechanism of superior neuroprotective effect of H2S-EVs.H2S modified MSCs up-regulated the expression of miR-7b-5p in H2S-EVs,H2S-EVs as the natural carrier of mir-7b-5p,delivered the rich content of mir-7b-5p to activated microglia/infiltrating mononuclear-macrophage in ipsilateral hemisphere after HI.Inhibition the activation of CD11b+/CD45low microglia and CDllb+/CD45high infiltrating mononuclear-macrophage activation and promotes M2 polarization,reduced neuroinflammation and thereby improving long-term learning and cognitive memory in HI mice.In addition,modification of EVs may also be a potential therapeutic strategy for perinatal brain injury.